Download Bacteroides mobilizable and conjugative genetic elements

Review
Carlos Quesada-Gómez1,2
Bacteroides mobilizable and conjugative
genetic elements: antibiotic resistance
among clinical isolates
1
2
Laboratorio de Investigación en Bacteriología Anaerobia, Centro de Investigación en Enfermedades Tropicales, Universidad de Costa Rica
Sección de Bacteriología General, Facultad de Microbiología, Universidad de Costa Rica.
ABSTRACT
The conjugation is one of the most important mechanisms
of horizontal gene transfer in prokaryotes, leading to genetic
variation within a species and the acquisition of new traits,
such as antibiotic resistance. Bacteroides is an obligate
anaerobe of the colon and a significant opportunistic
pathogen. Antibiotic resistance among Bacteroides spp. is
rapidly increasing, largely due to the dissemination of DNA
transfer factors (plasmids and transposons) harbored by
members of this genus. Transfer factors can be divided into two
classes, conjugative and mobilizable.
Species of the intestinal Bacteroides have yielded different
resistance plasmids, all of which have been intensely studied,
the plasmids encode high-level MLS resistance conferred by a
conserved erm gene.
It has been reported an interesting observation associated
with the transfer of several of these types of elements, all of
which conferred Tcr and displayed greatly increased transfer
efficiency following exposure to tetracycline. Many of the
conjugative transposons (CTns) in Bacteroides are related to
various genetic elements (such as CTnDOT, CTnERL, NBU and
others). CTnDOT carries a tetracycline resistance gene, tetQ, and
an erythromycin resistance gene, ermF.
Resistance to drugs used to treat Bacteroides infections,
such as clindamycin, has also been increasing. These conjugal
elements have been found in Bacteroides clinical isolates. Thus,
horizontal gene transfer could conceivably have played a role
in the rising incidence of resistance in this bacterial group.
Keywords: antimicrobial resistance, Bacteroides , conjugative
transposons
Correspondence:
Carlos Quesada-Gómez
Laboratorio de Investigación en Bacteriología
Anaerobia, Facultad de Microbiología, Universidad
de Costa Rica. 2060, San Pedro, Montes de Oca,
San José, Costa Rica.
E-mail: [email protected]
184
Elementos genéticos conjugativos y
mobilizables en Bacteroides: resistencia a los
antibióticos en aislamientos clínicos
RESUMEN
La conjugación es uno de los mecanismos más importantes
de la transferencia horizontal de genes en procariotas, lo que
lleva a la variación genética dentro de una especie y la
adquisición de nuevos rasgos, como la resistencia a los
antibióticos. Bacteroides es un anaerobio obligado y un
patógeno oportunista importante. La resistencia a los
antibióticos entre especies de Bacteroides está aumentando
rápidamente, debido en gran parte a la difusión de los factores
de transferencia de ADN (plásmidos y transposones) albergado
por los miembros de este género. Los factores de transferencia
se pueden dividir en dos clases, conjugativos y movilizables. Las
especies de Bacteroides han presentado plásmidos de
resistencia a los antibióticos, todos los cuales han sido
intensamente estudiados. Estos plásmidos codifican de alto
nivel de resistencia MLS conferida por un gen erm conservado.
Se ha informado una observación interesante asociada a la
transferencia de varios de estos tipos de elementos, todo lo cual
confiere mayor resistencia y que aparecen en gran medida la
eficiencia de transferencia después de la exposición a la
tetraciclina. Muchos de los transposones conjugativos (CTn) en
Bacteroides están relacionados con varios elementos genéticos
(como CTnDOT, CnTnERL, NBU y otros). CTnDOT lleva un gen de
resistencia a la tetraciclina, tetQ, y un gen de resistencia a la
eritromicina, ermF. La resistencia a los medicamentos utilizados
para tratar infecciones por Bacteroides, tales como la
clindamicina, también ha ido en aumento. Estos elementos
conjugativos han sido encontrados en los aislados clínicos de
Bacteroides. Por lo tanto, la transferencia horizontal de genes
posiblemente podría jugar un papel importante en la creciente
incidencia de la resistencia bacteriana en este grupo.
Palabras clave: resistencia antimicrobiana, Bacteroides, transposones conjungativos
Rev Esp Quimioter 2011;24 (4): 184-190
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C. Quesada-Gómez
Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates
INTRODUCTION
plasmids encode high-level MLS resistance conferred by a
conserved erm gene6,9,10.
Conjugal genetic exchange in Bacteroides was first
described in 1979 when reports from three laboratories
demonstrated conjugally transmissible macrolidelincosamide-streptogramin (MLS) resistance in this genus.
This resistance phenotype was important because it
permitted bacterial growth in the presence of clinically
effective and widely used antibiotic clindamycin. This
resistance phenotype was similar to that seen in Grampositive bacteria (e.g., streptococci, staphylococci and bacilli).
The nucleotide sequence of two of the erm genes (ermF
from pBF4 and ermFS from pBI136) has been determined,
and the genes have been found to differ at only one
nucleotide position. Physical analyses of both wild-type and
mutant plasmids revealed the erm gene of each plasmid was
bordered by a similar, if no identical, sequence, which flanked
the gene in a directly repeated fashion. Other than their erm
genes and the associated directly repeated DNA sequences,
pBF4, pBFTM10 and pBI136 shared no detectable homology11.
In these bacteria, the MLS gene (designated erm)
encoded and RNA methylase that modified specific adenine
residues in the 23S rRNA, thus preventing MLS drugs from
binding to the ribosome and inhibiting protein synthesis. The
genetic basis of such conjugal transfer has been traced to
both plasmids as well a nonplasmid elements. The latter
appear to reside chromosomally and to resemble the
conjugative transposons of the Gram-positive bacteria in
their behavior1.
The repeated sequences bordering the erm genes of
these three plasmids have been shown to be a related, if no
identical, insertion sequence (IS) elements (IS4400 on
pBFTM10, IS4351 on pBF4, and iso-IS4351 on pBI136). These
flanking IS elements form compound transposons with their
associated erm genes, and genetic evidence for their
transposition in both B. fragilis and Escherichia coli. In all
three of these transposons the erm genes were juxtaposed to
one copy of the IS element, suggesting some interaction
between these two sequences. This suggestion has been
experimentally explored by the analysis of the nucleotide
sequence data, S1 nuclease transcription mapping studies,
and the construction of gene fusions to evaluate promoter
activity. Collectively, these data indicate that the erm genes
arer under the transcriptional control of the promotors
contained in the IS element1,12,13.
The conjugation is one of the most important
mechanisms of horizontal gene transfer in prokaryotes,
leading to genetic variation within a species and the
acquisition of new traits, such as antibiotic resistance or the
ability to produce toxins. Bacteroides conjugative
transposons (CTn) were among the first transposons
discovered, and they have since been shown to play a central
role in the dissemination of antibiotic resistance genes in the
related genera2.
In particular, a family of CTns, exemplified by CTnDOT
and CTnERL, appears to be playing an important role in
transferring resistance genes among Bacteroides strains.
CTns of this family not only transfer themselves but also
mobilize coresident plasmids. In addition, proteins encoded
on these CTns trigger in trans the excision and circularization
of mobilizable integrated elements called NBUs, and they
mobilize these circular forms Bacteroides or Escherichia coli
recipients3.
They may also mobilize other integrated elements that
have been given transposon designation, such as Tn4399,
Tn4555, Tn55204,5. To distinguish mobilizable elements that
have been given a transposon designation from
nonmobilizable Bacteroides transposons such as Tn4351 or
Tn45516,7,8.
BACTEROIDES TRANSPOSONS IN ANTIBIOTIC
RESISTANCE PLASMIDS
Species of the intestinal Bacteroides have yielded three
different resistance plasmids, all of which have been
intensely studied: pBF4, a 41-kb plasmid from B. fragilis;
pBFTM10, a 15-kb plasmid from B. fragilis (and its
indistinguishable counterpart pCP1 from B.
thetaiotaomicron); and pBI136, an 80 kb-plasmid from B.
ovatus all share some common features (table 1). All three
25
Examples of the IS element being in opposite orientation
with respect to the erm gene were found in the comparative
examination of pBF4 and pBFTM10 with pBI136. Odelson et
al.10 have proposed a model to explain the formation of these
transposons tha predicts that all three transposons evolved
independently of one another. Following the entry of a
similar erm gene into Bacteroides, transposition of the IS
element next to the newly acquired gene resulted in the
transcriptional control of erm by the IS element. This may
have been required in the face of selective pressure because
the invading erm gene could not be expressed in Bacteroides
for several reasons (e.g., unrecognizable promoter,
nofunctional upstream regulatory sequences on the erm
mRNA)10.
A NEW NONPLASMID CONJUGATIVE ELEMENTS
IN BACTEROIDES
It has been know since early in the 1980s that certain
antibiotic-resistant strains of Bacteroides fragilis were
capable of transferring their resistance without the
involvement of detectable plasmid DNA. Such transfer
elements have been placed in two categories with respect to
antibiotic resistance phenotypes: those that transfer
tetracycline resistance alone and those which transfer
tetracycline and clindamycin resistances in a linked fashion9.
Salyers and Shoemaker 12 reported an interesting
observation associated with the transfer of several of these
Rev Esp Quimioter 2011;24 (4): 184-190
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C. Quesada-Gómez
Table 1
Genetic element
Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates
Summary of mobilizable and conjugative genetics elements that confers
antimicrobial resistance in Bacteroides.
Description
pBF4, pBFTM10, pBI136
41-kb, 15-kb, 80-kb plasmids. They encode high-level MLS resistance conferred by erm genes
Tn5030
ERL-type element. Mobilizable transposon. That encodes tetracycline and MLS resistance
Tn4399
Transposon capable of mobilizing plasmids to E. coli recipient.
CTnDOT
Major conjugative transposon from Bacteroides. That carries tetQ and ermF genes
Tn916
Enterococcal CTnDOT-type elements found in Bacteroides isolates
NBU
Non-replicating Bacteroides units. NBUs, like the CTn that excise and mobilize them, excise to form covalently closed circular intermediates
Tn5520, Tn4555
Mobilizable transposons, they rely on the CnTn for transfer. This confers resistance to other antibiotics (such as β-lactamics)
CTnBST
Conjugative transposon. 100 kbp. This contains ermB gene
CTn341
CTn of the CTnDOT family. Major functional genes: DNA metabolism, conjugation and antibiotic resistance.
cLV25
Movilizable transposon with similarity to the Tn4399
types of elements, all of which conferred Tcr and displayed
greatly increased transfer efficiency following exposure to
tetracycline. Specifically, when these elements were
transferred to B. uniformis and the resulting strain was
exposed to inhibitory tetracycline, two circular DNA forms
(10 and 11-kb) could be detected as extrachromosomal
elements. Southern analyses indicated these circular
molecules were normally integrated into the host cell
chromosome. Certain of the Tc r nonplasmid elements
examined by these authors did not mediate the excision of
circular DNA forms.
Stevens et al.13 have presented evidence suggesting that
the control of production of these circular DNA forms resides
in a region contained within the Tcr transfer element called
ERL (confers Tcr and MLS resistance). Halula and Macrina14
have also reported the partial cloning of Tcr, MLSr element,
called Tn5030, from B. fragilis. They estimated the size of this
element to be in the 40 to 50-kb range, based on overlapping
clones. Interpretation of their results was complicated by the
unexpected finding that sequences related to Tn5030 were
present in the transconjugant strain from wich their
genomic libraries were prepared. Further studies are needed
to fully define the molecular structure of this element.
Tn4399 was capable of mobilizing plasmids in
intergeneric Bacteroides matings as well as in Bacteroides-E.
coli matings (table 1). This transfer element provides yet
another novel example of the genetic plasticity in anaerobic
bacteria, and its is likely to play a role in the dissemination of
antibiotic resistance determinants2.
Other feature is the ability of most Bacteroides CTns to
induce conjugation 1000- to 10000-fold in the presence of
tetracycline. The transfer of both the CTn and unliked
elements is enhaced by growth in the presence tetracycline
at concentration of < 1 mg/L, and this induction is mediated
by a complex signal transduction pathway that includes a
two-component regulatory system (RteA and RteB) and at
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least one other regulatory protein, RteC 15. The ability to
mobilize other genetic elements and the antibioticstimulated transfer may in part explain the high tetracycline
resistance and the high frequency of the other antibiotic
resistance genes in Bacteroides16.
CTnDOT: A BACTEROIDES CONJUGATIVE TRANSPOSON
Many of the conjugative transposons (CTns) in Bacteroides
are related to an element calle CTnDOT (table 1). CTnDOT carries
a tetracycline resistance gene, tetQ, and an erythromycin
resistance gene, ermF16.
In fact, without tetracycline stimulation of donor cells,
virtually no transfer occurs. Any studies identified a central
regulatory region on CTnDOT that is required for tetracycline
induction of transfer functions. This region contains a threegene operon that consists of the tetracycline resistance gene
tetQ and two regulatory genes, rteA and rteB.
RteA and RteB are most closely related to the sensor and
response regulator components, respectively, of twocomponent regulatory systems. RteA presumably activates
RteB, and activated RteB stimulates the expression of excision
and transfer genes.
The regulatory cascade mediated by RteA, RteB and RteC is
not, however, the first step in tetracycline stimulation of CTn
transfer in Bacteroides. Expression of the tetQ-rteA-rteB
operon itself is controlled by tetracycline. Stevens et al.13
reported that fusion of tetQ to the reporter gene uidA
produced fusion whose expression was stimulated by
tetracycline.
Intercellular transposition of CTns in Bacteroides is
thought to occur by the following steps. The CTn initiates
conjugal transfer by excising from the chromosome to form a
circular intermediate. This intermediate is nicked at the transfer
origin (oriT), and a single-strand copy is transferred to the
Rev Esp Quimioter 2011;24 (4): 184-190
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C. Quesada-Gómez
Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates
recipient cell, recircularized, and integrated into the recipient´s
genome15,16.
Since CTnDOT appeared to integrate site selectively, the
comparions of the sequences of the ends of CTnDOT with the
sequences of the integration sites revealed some sequence
similarity. One candidate for recognition sequence is a 10-bp
sequence that is immediately adjacent to the site where
CTnDOT entered the cromosome and at the right end of the
circular form of the element. In the integrated form, this target
10-bp sequenced is 5-bp from the left end of CTnDOT from
insertions in four other chromosomal sites. Similar 10-bp
sequences were seen in all four cases7,15,16.
The integration and excision of CTns have been studied in
detail only in the case of Tn916, a CTn first found in
enterococci. The CTnDOT-type elements differ in a number of
ways from Tn916. First, Tn916 integrates relatively randomly
whereas CTnDOT appears to integrate site selectively into about
seven sites on the Bacteroides chromosome. Second, there is no
sequence similarity between CTnDOT and Tn916 in any of the
regions so far sequenced. A third difference between CTnDOT
and Tn916 is that transfer of CTnDOT is stimulated 1000- to
10000-fold by tetracycline, a fold increase much higher than
the tetracycline stimulation reported for Tn916 transfer8.
The tetracycline-dependet stimulation of CTnDOT transfer
is mediated by proteins encoded by three regulatory genes:
rteA, rteB and rteC. No regulatory genes analogous to the rte
genes of CTnDOT have been found on Tn91616.
REGULATION OF BACTEROIDES OPERON THAT
CONTROLS EXCISION AND TRANSFER OF THE
CTNDOT
Exist a translational attenuation-type regulation of the
tetQ-rteA-rteB operon in response to tetracycline. RT-PCR
analysis showed that similar amount of the mRNA from this
operon is made regardless of whether tetracycline is present
result rules out another possible model for attenuational
control of gene expression, the binding of tetracycline to a
structure in the mRNA itself in such a way as to cause
antitermination.
In the case of an antitermination type of control, the
concentration of tetQ mRNA should have been much higher
under inducing conditions. Although tetQ mRNA was made
both in the presence and absence of tetracycline stimulation,
translation of the message, is much higher in tetracyclinestimulated cells than in cells no stimulated. This observation
taken together with the fact that a translational fusion with
uidA was responsible for much higher activity and a higher
increase in tetracycline-stimulated cella than a transcriptional
fusion in the same locus8, supports the idea that regulation
occurs at the level of translation rather than transcriptional.
Further evidence for translational attenuation came from
mutagenesis experiments that were guided but the observation
that hairpain structure could form in the leader region of the
mRNA.
27
Production of RteA and RteB, the other protein encoded in
the tetQ-rteA-rteB operon, should be regulated similarly to
production of TetQ. That is, the proteins are produce at detectable
levels only when the cells are stimulated with tetracycline. Since
rteA and rteB have been shown to be in the same operon as tetQ,
production of these proteins is presumably due to translational
coupling one ribosomes begin to translate the operon mRNA.
RteA and RteB have no role in tetracycline regulation of operon
expression. This was somewhat surprising because RteA and RteB
are both essential for CTn excision and transfer functions and
resemble components of a two-component regulatory system.
RteA, the putative sensor protein, is a sensing something other
than tetracycline15,16.
TetQ was not required for tetracycline stimulation, but its
presence did have an effect. Moreover, increased levels of TetQ
due increased levels copy numbers of tetQ caused a greater
decrease in protein production. TetQ is a ribosome protection
type of tetracycline resistance protein, which is most closely
related to an elongation factor. TetQ modifies ribosomes so
that tetracycline no longer binds as efficiently. The fact that a
higher intracellular concentration of TetQ increased the MIC
and further decreased the translation of operon mRNA suggets
that one copy of tetQ is no sufficient to protect all of the
ribosomes in the cell 8. The modulating effect of TetQ on
translation may be a means for the cell to limit the production
of RteA and RteB and thus the amount of CTn excision and
transfer that occurs. Excision of CTnDOT in Bacteroides is
apparently not a lethal event because the chromosomal site
from which the CTn is excised is resealed in the process16, but
too much activity of this sort could have some deleterious
effect on fitness that could be important in an enviroment as
competitive as the human colon.
THE NBUs ELEMENTS
Bacteroides conjugative transposons can also mediate the
excision and mobilization of unliked integrated elements called
NBUs (non-replicating Bacteroides units). The NBUs so far
characterized are 10 to 12 kbp in size. NBUs, like the
conjugative transposons that excise and mobilize them, excise
to form covalently closed circular intermediates that are
transferred by conjugation to a recipient, where their integrate
into recipient genome17 (table 1).
The circular forms of the NBUs appeared not replicate and
were designated nonreplicating units to distinguish them for
the plasmids. The circular forms of the NBUs were see only
when the strain was grown in medium containing tetracycline.
Subsequently, we found that the NBUs are normally integrated
in the chromosome and that trans action by a Tcr element is
requerired to excise and circularize them18.
The NBUs are sometimes cotransferred with Tcr elements,
and it was postulated that the excised circular form of the
NBUs were plasmidlike forms and were transferred like
plasmids and the integrated into the recipient chrosomome3,17.
The mobilizable element NBU2 (11 kbp) was found
Rev Esp Quimioter 2011;24 (4): 184-190
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C. Quesada-Gómez
Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates
originally in two Bacteroides clinical isolated, B. fragilis ERL and
B. thethaiotamicron DOT. NBU2 appeared to be very similar to
NBU1 in a 2,5 kbp internal region, but this identify between
NBU1 and NBU2 is limited to this small region. The sequence of
NBU2 revealed two ORFs whose derived amino acid sequences
were related to those of known antibiotic resistance genes
previously found in Gram-positive bacteria. The deduced amino
acid sequence of linAN2, LinA(N2), had 50 to 52% identify and
70 to 72% similitary to the LinA´of Staphylococcus aureus and
LinA on pIP855 in S. haemolyticus. LinA is an Onucleotidyltransferase which inactives lincosamides including
lincomycin and clindamycin. This is also the first sighting of a
linA type gene in Bacteroides species3,18.
TN4555 AND THE ANTIBIOTIC RESISTANCE IN
BACTEROIDES
In Bacteroides species are integrated elements that are
much smaller than the CTns. These elements, exemplified by
NBU1, Tn5520 and Tn4555, have been called mobilizable
transposons because they rely on the CTns for transfer
functions8 (table 1).
The integrases of NBU1, Tn5520 and Tn4555 have been
sequenced, and all are members of the lambda family
integrases. NBU1 excision more closely resembles excision of
phage lambda in that there is an att site formed by the joined
ends that integrates into an identical site in the choromosome5.
Although the mobilizable transposons rely absolutely on the
CTns for transfer, there is so far no evidence for any significant
sequence similarity between them and the CTns.
Bacteroides are potencial reservoirs of antibiotic
resistance genes and Tn4555 from Bacteroides vulgatus carries
cfxA, encoding an extended spectrum ß-lactamase found in
80% of cefoxitin-resistant, imipenem-sensitive Bacteroides
species tested19.
Cefoxitin resistance was observed shortly after its
introduction in the 1970s and elements such as Tn4555 may be
responsible for the rapid emergence of resistance. More
recently, a ß-lactamase gene 98% identical to cfxA was found
in 97% of amoxicillin-resistant Prevotella isolated from
periodontitis patients20. Since there is potential for Tn4555
and like elements to undermine the effectiveness of antibiotic
treatment, it is important to understand all aspects of their
dissemination.
The mobilizable transposon (MTn), Tn4555 can be
transferred (mobilized) to other bacteria when in the presence
of CTn, a self-transmissible genetic element that moves from
donor genome to recipient genome during cell to cell contact.
Although transposons have been defined as mobile genetic
elements that randomly insert into DNA by mechanisms not
requiring homologous recombination, all know elements
including MTns and CTns show some degree of target site
selectivity8.
CTns appear to have unique target site selection
mechanisms which in some cases require homology between
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the CTn ends (attTn) and the tartget. For example, CTnDOT
targets a 10-bp sequence in the Bacteroides choromosome
that has homology to its right end15.
Tn4555, a clinically relevant MTn, carries all the genes
necessary for its insertion into phylogenetically diverse hots. In
the investigation of Bacic and Smith4 all bacteria tested, there
was preferred insertion site or target and while there were
some shared motifs, none of these had ends. A lack of
homology suggested that sequence alone was not the main
determining factor for site selection, though the translated
Bacteroides targets did have homology to BspA from B.
forsythus. BspA is a cell-surface-associated, leucine-rich
protein involved in adhesion to fibronectin and fibrinogen.
BspA has repetitive amino acid sequences suggesting that
repetitive areas in the DNA sequence are important for large,
imperfect, direct repeats; however, there were no significant
repeats in the other Bacteroides targets nor in the E. coli target,
and the E. coli target was highly preferred with 90% of all
insertions occurring there, more than for any of the others.
Also the site of Tn4555 insertion in the original clinical isolate
B. vulgatus had no repeats nor did it have any amino acid
homology to BspA8.
The Mtn share a functional similarity to mobilizable
plasmids in that they are not self-transmissible but rather they
encode an element-specific mobilization gene(s) and a transfer
origin, oriT, that utilize Tcr-element conjugative apparatus to
effect their transfer to a new host. These mobilization genes
appear to be primarily responsible for DNA processing reactions
in which they catalyze a strand-specific nick in the transposon
at oriT15.
Tn4555 is a mobilizable transposon that carries the cfxA
gene, which encodes a clinically significant, broad spectrum ßlactamase responsible for widespread resistance to cefoxitin
and other ß-lactams. Initial studies on Tn4555 integration
indicated that it proceeds by a site-specific recombination
mechanism that utilizes a 6-bp crossover region in the target
site20. Clearly Tn4555 is not limited to one or two Bacteroides
species or genera in the intestinal niche. There was no
difference in excision from preferred sites versus non-preferred
sites therefore these targets must benefit insertion5,8.
Tn4555 carries antibiotic resistance genes and these
elements can be mobilized by a variety of genetic elements can
be mobilized by a variety of genetic elements including large Rplasmids or by conjugal transposons such as the large
Bacteroides Tc r -elements 5 . However, unlike mobilizable
plasmids, there transposons have minimal requerimets for
stable maintenance in new hosts.
OTHERS CONJUGATIVE TRANSPOSONS
Gupta et al.21 used pulsed-field gel electrophoresis to show
that the B. uniformis element carrying ermB was integrated
into the chromosome and was thus probably a CTn (table 1).
The CTn was named CTnBST. CTnBST was estimated to be about
100 kbp in size. A 13-kbp region containing ermB was
Rev Esp Quimioter 2011;24 (4): 184-190
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C. Quesada-Gómez
Bacteroides mobilizable and conjugative genetic elements: antibiotic resistance among clinical isolates
sequenced and compared to sequences then in the databases.
The DNA sequence of the ermB gene was identical to genes
found in Clostridium difficile. Outside the ermB gene, however,
only a few hundred base pairs downstream of ermB had high
sequence identity to DNA from gram-positive bacteria22.
A Bacteroides CTn, CTnBST, integrates more site
specifically than other well-studied CTns, CTnDOT and the
enterococcal CTn Tn916. Moreover, the integrase of CTnBST,
IntBST, had the C-terminal 6-amino-acid signature that is
associated with the catalytic regions of members of the
tyrosine recombinase family, most of which integrate site
specifically (table 1). Also, in most of these integrases, all of the
conserved amino acids are required for integration23. However,
the IntBST had that changing three of the six conserved amino
acids in the signature, one of which was the presumed catalytic
tyrosine, resulted in a 1,000-fold decrease in integration
frequency. Also, CTnBST differed from CTnDOT in that whereas
the transfer frequency of CTnDOT is stimulated 100- to 1,000fold by tetracycline, CTnBST exhibited constitutive transfer24.
CTn341, originally found in a clinical isolate of Bacteroides
vulgatus, is 52 kb in size and encodes genes for tetracycline
resistance and regulation of transfer, conjugation, and DNA
metabolism. The mob region of CTn341 is comprised of three
genes, mobABC , as well as the transfer origin, oriT. The oriT
region of CTn341 has been located within 100 bp of mobA and
the putative nic site was identified within this region. The Mob
proteins are predicted to bind at this site and form the
relaxosome complex which specifically nicks the DNA at the
nick site prior to coupling it to the mating pore apparatus25. The
mob gene expression is tightly regulated and transcription is
induced by low levels of tetracycline through the two
component regulatory system genes rteA-rteB.
CTn341, is a member of the most common CTn group, the
CTnDOT family26. Sequence analysis revealed 46 genes and one
functional group II intron (table 1). The genes fell roughly into
three major functional groups: DNA metabolism, regulation
and antibiotic resistance, and conjugation 27 . Mutational
analyses of genes from each group were used to verify the
proposed functional assignments. Based on G+C content and
codon usage, the functional groups appeared to belong to
different genetic lineages, indicating that CTn341 is a
composite, modular element. Comparisons with Bacteroides
genome sequences suggested that the basic conjugation and
excision genes are conserved in Bacteroides spp27.
Other transfer factor is the cLV25. The cLV25 mobilization
proteins have similarity to the Tn4399 mobilization proteins
and to mobilization proteins encoded by the CTnDOT ermF
region. In addition, the genetic organization of the cLV25
mobilization genes and oriT is analogous to those of Tn4399
and the CTnDOT ermF region28 (table 1). Further, cLV25 can
functionally crossreact with Tn4399. The cLV25 is a mobilizable
transposon based on the presence of imperfect inverted
repeats at its termini, an apparent target site repeat, and an
open reading frame whose predicted protein has similarity to
integrases28.
29
CONCLUSIONS
In general, Bacteroides are resistant to a wide variety of
(recently many species have acquired resistance to
erythromycin, clindamycin, and tetracycline). This high level of
antibiotic resistance has prompted concerns that Bacteroides
species may become a reservoir for resistance in other, more
highly-pathogenic bacterial strains.
The conjugation is one of the most important mechanisms
in the horizontal transfer of genes in prokaryotes, providing the
capacity of variation between species by means of the
obtaining of new genetic information.
To beginnings of the years 80s the scientific community
demonstrated that certain clinical strains of B. fragilis resistant
to the antibiotics were capable of transmitting this resistance
by means of plasmids and without the presence of these. These
elements establish two categories with regard to the resistance
of antibiotics: the transfer of the resistance to tetracycline and
the transfer of resistance to tetracycline and clindamycin
together.
The CTn in Bacteroides that can manage to join to the
chromosome or plasmids of the receptor bacteria. The potential
of the CTn in Bacteroides due to the fact that they are capable
of mobilizing plasmids co-residents in trans and in cis, and also
of stimulating the excision and the transfer of integrated
elements called units not replicate of Bacteroides.
The family of the CTn they are represented by CTnDOT and
CTnERL, which increase the rates of conjugation in presence of
1 mg/L of tetracycline, which is very significant for that this
antibiotic not only would select Bacteroides's resistant strains
if not that in addition would stimulate the transfer of genetic
elements and of genes of resistance between bacteria y this
situation could complicate the antimicrobial treatment in a
several Bacteroides human infections.
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